The invention relates to a device for thermocycling polyelectrolytes, in particular for amplification of nucleic acids. Specifically, the invention involves trapping minute amounts of nucleic acids in a microfabricated, dielectrically focused device, thermocycling them, and releasing them for fractionation or analysis.
The amplification of nucleic acids is central to the current field of molecular biology. Library screening, cloning, forensic analysis, genetic disease screening and other increasingly powerful techniques rely on the amplification of extremely small amounts of nucleic acids. As these techniques are reduced to a smaller scale for individual samples, the number of different samples that can be processed automatically in one assay expands dramatically. For further improvements, new integrated approaches for the handling and assaying of a large number of small samples are needed.
With the polymerase chain reaction (PCR) for nucleic acid amplification, a purified DNA polymerase enzyme is used to replicate the sample DNA in vitro. This system uses a set of at least two primers complementary to each strand of the sample nucleic acid template. Initially, the sample nucleic acid is heated to cause denaturation to single strands, followed by annealing of the primers to the single strands, at a lower temperature. The temperature is then adjusted to allow for extension of the primers by the polymerase along the template, thus replicating the strands. Subsequent thermal cycles repeat the denaturing, annealing and extending steps, which results in an exponential accumulation of replicated nucleic acid products.
PCR represents a considerable time savings over the replication of plasmid DNA in bacteria, but it still requires several hours. PCR also has limitations in the subsequent handling of the product. Most reactions occur isolated in a test tube or plate containing the required reagents. Further analysis of these products entails removing them from the tube and aliquoting to a new environment. Significant delay and loss and damage to the product may result from such a transfer. Emerging technology in the display of nucleic acids in arrays on chips, for further identification and selection, requires a more precise method of transfer of samples from amplification step to chip than is possible by dispensing the contents of each reaction tube individually.
Another disadvantage of PCR is the requirement of a reaction tube which has a volume possibly too large for the amplification of particularly minute amounts of starting template and other reagents. PCR is able to amplify just one molecule of template, but the volume of the reaction mixture makes this goal difficult to achieve. The nature of the reaction tube also requires a significant volume of the other reagents. Conventional PCR involves heating and cooling the reaction tube several times for each cycle, requiring elaborate instrumentation to control the temperature of the apparatus which holds the tubes, and the tubes and solutions they contain, over time.
Lab-on-a-chip or biochip technology for manipulating DNA on a small scale is a recent development in the art. For example, Austin et al., U.S. Pat. No. 5,427,663, describes a microlithographic array for fractionating macromolecules. Heller, U.S. Pat. No. 5,605,662 describes a microfabricated device having DC microelectrodes for DNA hybridization. Individual wires in a direct electrophoretic field have been used to focus and launch DNA into separation media.
Microfabricated devices can be used for PCR. Wilding, et al. (Clin.Chem. 40:1815-8, 1994) designed a photolithographed, sealed silicon chip which can receive reagents by capillary action and can be mounted on a Peltier heater-cooler. This design reduces reagent volumes, but requires an external source for heating and does not couple positioning and manipulation of the nucleic acids, and so amplification and subsequent analysis on a fractionation matrix cannot be achieved without transfer of samples. Thus, prior art systems for the amplification of nucleic acids do not integrate micromanipulation and amplification steps, such that the need for transfer steps reduces the quantity and quality of the products, and time and labor are increased.
In another field, dielectrophoresis has been used to position cells and molecules on a micron scale. Washizu and Kurosawa, IEEE Transactions on Industry Applications, 26 (6): 1165-1172, 1990, and Washizu et al., IEEE Transactions and Industry Application, 31(3):447-455, 1995, used high voltage (10.sup.4 V/cm) and high frequency (1 MHz) alternating currents to generate a dielectric field in a micron-sized floating electrode. Alignment, permanent fixation, stretching, and cutting of DNA molecules was described. Asbury and van den Engh (Biophys. J. 74:1024-30, 1998) report a sealed device having an array of 100 strips of gold which, when placed in an oscillating field, reversibly traps DNA along the edges of the strips. These papers did not demonstrate fractionation of DNAs, and do not suggest dielectric trapping in a thermal cycling device for DNA amplification. Such devices are unsuitable for complex enzymatic reactions, such as PCR, which require precise temperature control, and do not provide microfluidic structures that can be used for transfer to a fractionating medium without damage. Moreover, as the Takashima group claimed in the 1970s, the dielectric constant of single stranded DNA is less than double stranded DNA which would discourage use of dielectrophoresis for single stranded uses such as PCR.